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NEUROPHARMACOLOGY
Neuroscience Drug Discovery, Movement Disorders, Merck Research Laboratories, West Point, Pennsylvania (O.V., G.R.S., M.J.M.); Department of Pharmacology, University of Florence, Florence, Italy (G.M.); and Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (P.J.C.)
Received November 10, 2004; accepted March 3, 2005.
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Abstract |
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,b; Tepper et al., 1995). The finding that SNc neurons recorded in brain slice preparations only exhibit pacemaker-like activity has highlighted the importance of afferent inputs in determining the firing pattern of these cells. Anatomical and electrophysiological evidence exists for a direct excitatory projection from the subthalamic nucleus (STN) to the dopaminergic neurons in the SNc (Smith et al., 1990; Smith and Grace, 1992; Iribe et al., 1999) that may in part modulate SNc bursting (Smith and Grace, 1992). This glutamatergic input has been the focus of much attention based on the findings that the STN is overactive in Parkinson's disease (PD) and that glutamate is believed to play a role in the continued degeneration of the SNc dopamine neurons in this disorder (Rodriguez et al., 1998); however, relatively little work has been focused on the modulatory control of these excitatory inputs.
The metabotropic glutamate receptors (mGluRs) are a class of G-protein coupled receptors that play a variety of neuromodulatory roles in the central nervous system. To date, eight mGluR subtypes have been cloned (for review, see Conn and Pin, 1997). The mGluRs are classified into three groups based on sequence homology, agonist and antagonist pharmacology, and coupling to signal transduction pathways in expression systems. Group I (mGluR1 and mGluR5) mGluRs are preferentially expressed in postsynaptic region, where they modulate cell excitability. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7, and mGluR8) mGluRs are presynaptically localized and modulate the release of neurotransmitters. Based on the localization in the basal ganglia motor loop and the functional roles that have been revealed to date, the group III mGluRs, and mGluR4 in particular, hold promise for the design of novel palliative treatments for PD (Marino et al., 2003a).
We previously have shown that presynaptically localized mGluR4 modulates inhibitory transmission at the striatopallidal synapse, a critical synapse in the indirect pathway of the basal ganglia motor system (Valenti et al., 2003). Furthermore, when studied in both acute and chronic behavioral models of PD, activation of mGluR4 produces a dramatic reversal of motor impairment, suggesting mGluR4 may provide a potential target for the palliative treatment of PD (Valenti et al., 2003). Recently, Wigmore and Lacey (1998) have shown that the activation of group III mGluRs inhibits excitatory transmission onto midbrain dopamine neurons. Recent developments in selective pharmacological tools (Thomas et al., 2001; Flor et al., 2002; Marino et al., 2003b) and the availability of knockout mice make it possible to determine with a high degree of certainty which group III mGluR mediates a particular physiological effect. Based on the potential role of excitatory inputs to the SNc in determining the normal physiology of these cells, as well as the neurodegeneration associated with PD, we set out to pharmacologically characterize this group III mGluR-mediated modulation of transmission.
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Materials and Methods |
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Animals. All animals used in these studies were cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Merck Research Laboratories Institutional Animal Care and Use Committee (IACUC) approved all studies described in this study, and experimental protocols were in accordance with all applicable guidelines regarding the care and use of animals. Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AALAC) International approved facility with free access to food and water.
Slice Preparation. All electrophysiology experiments were performed on normal parasagittal slices from either 15- to 20-day-old Sprague-Dawley rats (Taconic Farms, Germantown, NY) or 7- to 12-day-old mice. The mGluR4 knockout mice (Gprc1d) (Pekhletski et al., 1996) were maintained in a colony at Emory University (Atlanta, GA). Wild-type 129X1/SvJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were killed by decapitation, and brains were rapidly removed and submerged in an ice-cold choline replacement solution containing 126 mM choline chloride, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.3 mM MgCl2, 8 mM MgSO4, 10 mM glucose, and 26 mM NaHCO3, equilibrated with 95% O2/5% CO2. The brain was glued to the chuck of a vibrating blade microtome (Leica Microsystems Nussloch GmbH, Nussloch, Germany), and 250-µm thick slices were obtained. Slices were immediately transferred to a holding chamber containing normal artificial cerebrospinal fluid (ACSF): 124 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2 mM CaCl2, 20 mM glucose, and 26 mM NaHCO3, equilibrated with 95% O2/5% CO2 that was maintained at room temperature. In all experiments, 5 µM glutathione, 500 µM pyruvate, and 250 µM kynurenic acid were included in the choline chloride buffer and in the holding chamber ACSF.
Electrophysiology. Whole-cell patch-clamp recordings were obtained as described previously (Valenti et al., 2003). During recording, slices were maintained fully submerged on the stage of a brain slice chamber at room temperature and perfused continuously with equilibrated ACSF (2–3 ml/min). Neurons were visualized using a differential interference contrast microscope and an infrared video system. Patch electrodes were pulled from borosilicate glass on a two-stage puller and had resistances in the range of 3 to 7 M
when filled with internal solution. To characterize the electrophysiological proprieties of the neurons, a potassium gluconate internal solution was used: 125 mM potassium gluconate, 4 mM NaCl, 6 mM NaH2PO4, 1 mM CaCl2, 2 mM MgSO4, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetrapotassium salt, 10 mM HEPES, 2 mM Mg-ATP, and 0.3 mM Na2-GTP. For all voltage-clamp experiments, the internal solution consisted of 140 mM cesium methane sulfonate, 16 mM HEPES, 10 mM NaCl, 2 mM EGTA, 2 mM Mg-ATP, and 0.2 mM Na2-GTP.
All recordings were done using HEKA EPC9 patch-clamp amplifiers (HEKA Elektronik, Lambrecht/Pfalz, Germany). Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation in the presence of blockers of GABAA (25 µM bicuculline methobromide) and GABAB (100 nM CGP55845 receptors. Bipolar tungsten stimulation electrodes were placed either locally in the SNc 
100 µm rostral to the recording site or in the STN. No difference was observed between these two groups, and the data were combined. EPSCs were evoked from a holding potential of -70 mV by single pulses that ranged from 3 to 15 V, 200 to 400 µs, delivered once every 30 to 60 s. Monosynaptic EPSCs for failure analysis were stimulated locally through a patch pipette filled with ACSF. These parameters were varied to optimize EPSC amplitude and stability. The SNc was recognized as a cell-dense region dorsal to the SNr in slices corresponding to Figs. 82 to 85 of the Paxinos and Watson rat brain atlas (1998) and Figs. 108 to 117 of the Paxinos and Franklin mouse brain atlas (2001).
All compounds were typically made in a 1000x stock and diluted into the ACSF immediately before use. L-AP4 and (S)-3,4-DCPG were made daily; all other compounds were aliquoted and stored at -20°C. Compounds were applied to the bath using a three-way stopcock. Agonists were always applied for 10 min to achieve a plateau concentration, whereas antagonists were applied 5 min prior to agonists and maintained in the bath during agonist application.
Statistics. Individual comparisons were made using a Student's or paired Student's t test as appropriate. In cases where multiple comparisons were evaluated, an ANOVA was employed followed by post hoc testing with Fisher's LSD test. All data are reported as mean ± standard error of the mean.
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Results |
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), we observed a homogeneous population of neurons with distinct electrophysiological properties, including low-frequency spike firing, large afterhyperpolarizations, and a prominent time-dependent inward rectification produced by the injection of hyperpolarizing currents (Fig. 1A).
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EPSCs were evoked as described in Materials and Methods. SNc dopaminergic neurons receive glutamatergic projections from the STN as well as a mix of glutamatergic and cholinergic inputs from the pedunculopontine nucleus (Lavoie and Parent, 1994; Charara et al., 1996). We attempted to favor the glutamatergic inputs from the STN by stimulating rostral to the recording site or within the STN. Single-shock electrical stimulation elicited a fast inward current, showing a short and constant latency that attained peak amplitude within approximately 10 ms (Fig. 1B). The glutamatergic nature of these EPSCs was confirmed by bath application of the AMPA/kainate-selective antagonist CNQX (20 µM). CNQX produced a complete block of the synaptic response (predrug amplitude: -260.94 ± 56.37 pA, mean ± S.E.M.; 20 µM CNQX amplitude: -19.98 ± 5.14 pA, mean ± S.E.M.; n = 5; p < 0.05, paired t test) (Fig. 1, B and D), suggesting that under these conditions these EPSCs are glutamatergic and predominantly due to activation of AMPA or kainate receptors.
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) have previously shown that the activation of group III mGluRs caused inhibition of excitatory postsynaptic potentials in a dose-dependent manner. This depression of the excitatory postsynaptic potentials was reversible and evident at a low concentration (0.3–30 µM) in a range that suggested activation of high-affinity receptors. We employed voltage-clamp methods to confirm that activation of group III mGluRs modulates excitatory transmission in midbrain dopamine neurons. The application of the highly selective group III mGluR agonist L-AP4 (Bushell et al., 1995) produced a significant inhibition of the evoked EPSCs (Fig. 2A) that reversed as the compound washed out of the bath (Fig. 2B). The response to L-AP4 was dose-dependent (Fig. 2C), with L-AP4 producing a maximal effect of 61.85 ± 2.82% inhibition at 3 µM (predrug amplitude: -204.2 ± 34.31 pA, mean ± S.E.M.; 3 µM L-AP4 amplitude: -82.73 ± 19.35 pA, mean ± S.E.M.; n = 7; p < 0.05, paired t test). L-AP4 exhibits potencies at recombinant rat group III mGluRs of 0.2 to 1 µM at mGluR4, 0.6 to 0.9 µM at mGluR6, 160 to 1300 µM at mGluR7, and 0.7 to 0.9 µM at mGluR8 (Schoepp et al., 1999). Since mGluR7 is only activated by high concentrations of L-AP4, and mGluR6 is not expressed at high levels in the brain (Nakajima et al., 1993), this effect is consistent with an action at either mGluR4 or mGluR8.
L-AP4 Inhibits Transmission by a Presynaptic Mechanism. Immunocytochemical studies have not detected significant levels of mGluR4 or mGluR8 in the SNc; however, in situ hybridization studies have reported low to moderate levels of mGluR4 and mGluR8 expression in both the SNc and the STN (Testa et al., 1994; Messenger et al., 2002). In many brain regions, the group III mGluRs are found presynaptically localized, where they modulate the release of neurotransmitter. Based on this, we would hypothesize that either mGluR4 or mGluR8 trafficked to the presynaptic STN terminals may mediate the L-AP4-induced inhibition of excitatory transmission in the SNc. To test this hypothesis, we attempted to determine whether L-AP4 reduces excitatory transmission in the SNc by a prior postsynaptic mechanism. We first attempted to study the effect of L-AP4 on paired-pulse facilitation. In these studies, we observed a trend toward an increase in paired-pulse ratio in three out of four cells; however, a high degree of variability in the baseline paired-pulse ratio prohibited an accurate quantitative analysis of these data. We next attempted an analysis of tetrodotoxin-resistant miniature EPSCs; however, the extremely low frequency of miniature EPSCs in this region made these studies impossible. Therefore, we employed an analysis of the effects of L-AP4 on failure of monosynaptic EPSCs (Malinow and Tsien, 1990). As described in Materials and Methods, focal electrical stimuli were employed to evoke single-fiber monosynaptic EPSCs that had a measurable failure rate (Fig. 3, A and B) (event probability: 0.79 ± 0.02; n = 4). In contrast to the evoked EPSCs described above, which are produced by the synchronized release of transmitter from many synapses, these events are monosynaptic. Thus, alterations in failure rate reflect changes in presynaptic release probability, whereas alterations in amplitude reflect changes in postsynaptic receptor function. Application of 3 µM L-AP4 produced a significant increase in failures (L-AP4 event probability: 0.13 ± 0.08; n = 4; p < 0.001, paired t test) (Fig. 3C). The failure rate increased in all cells studied; however, in one cell, the failure rate increased to 100%. Application of 3 µM L-AP4 still produced a significant increase in failure rate when this nonresponding cell was excluded from the analysis (predrug event probability: 0.80 ± 0.03, L-AP4 event probability: 0.18 ± 0.1; n = 3; p < 0.05, paired t test). When the mean amplitude of events from these three cells was compared with the predrug amplitude, no significant effect was observed (Fig. 3D) (predrug amplitude: -21.0 ± 3.1 pA, mean ± S.E.M.; 3 µM L-AP4 amplitude: -14.8 ± 1.2 pA, mean ± S.E.M.; n = 3; p > 0.1, paired t test). This increase in failure rate without effect on mean amplitude is consistent with a presynaptic site of action (Malinow and Tsien, 1990), suggesting that L-AP4 decreases excitatory transmission through a presynaptic mechanism of action.
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Pharmacology of the L-AP4 Mediated Inhibition of Excitatory Transmission in the Rat SNc. As discussed above, the different potency of L-AP4 at group III receptors suggests that the group III mGluR-mediated inhibition of excitatory transmission in the SNc is consistent with mGluR4 or mGluR8. Since mRNA for both mGluR4 and mGluR8 are expressed in the STN, the likely source of the glutamatergic afferents stimulated in these studies, it is possible that either receptor mediates this effect. We employed available pharmacological tools to attempt to distinguish between these two receptors. CPPG, a group III mGluR-preferring antagonist (Toms et al., 1996) was employed to confirm that this effect of L-AP4 is mediated by group III mGluRs. Preapplication of 100 µM CPPG inhibited the L-AP4-induced suppression of excitatory transmission in SNc neurons (predrug amplitude: -193.54 ± 29.3 pA, mean ± S.E.M.; 100 µM CPPG amplitude: -175.6 ± 34.3 pA, mean ± S.E.M.; n = 5; p > 0.05, paired t test) (Fig. 4C). To test for the involvement of mGluR8, we employed the mGluR8-selective agonist (S)-3,4-DCPG. (S)-3,4-DCPG exhibits potencies at recombinant human group III mGluRs of 8.8 µM at mGluR4, 3.6 µM at mGluR6, >100 µM at mGluR7, and 31 nM at mGluR8 (Thomas et al., 2001). Application of 300 nM (S)-3,4-DCPG, a concentration 10-fold higher than the EC50 of this compound at recombinant mGluR8, produced no effect on the excitatory transmission at SNc (predrug amplitude: -196 ± 14.7 pA, mean ± S.E.M.; 300 nM (S)-3,4-DCPG amplitude: -184 ± 7.1 pA, mean ± S.E.M.; n = 3; p > 0.1, paired t test) (Fig. 4, A–C). This result suggests that mGluR8 does not play a role in modulating transmission at the rat STN-SNc synapse; therefore, we hypothesized that mGluR4 mediates the effect of L-AP4 in modulating excitatory transmission onto dopaminergic neurons in the SNc. To test this hypothesis, we employed the recently characterized highly selective allosteric potentiator of mGluR4, PHCCC (Flor et al., 2002; Marino et al., 2003b). PHCCC potentiates recombinant rat mGluR4 with an EC50 of 3.7 µM and exhibits antagonist actions at all other group III mGluRs (Marino et al., 2003b). Therefore, PHCCC represents a selective and novel tool for studying the physiology of mGluR4. We employed PHCCC to confirm the hypothesis that mGluR4 modulates excitatory transmission in SNc. We choose a submaximal 300-nM dose of L-AP4 based on our dose-response study. Application of 300 nM L-AP4 produces a small yet significant reduction in excitatory transmission (predrug amplitude: -164.4 ± 32.2 pA, mean ± S.E.M.; 300 nM L-AP4 amplitude: -120.85 ± 24.57 pA, mean ± S.E.M.; n = 9; p < 0.05, paired t test). Application of the vehicle 1% dimethyl sulfoxide (data not shown) or 30 µM PHCCC alone had no effect on the subthalamo-nigral transmission (Fig. 5C). However, when coapplied with 300 nM L-AP4, 30 µM PHCCC produced a significant potentiation of the L-AP4-induced inhibition of the excitatory transmission (predrug amplitude: -208.83 ± 68.36 pA, mean ± S.E.M.; 300 nM L-AP4 + 30 µM PHCCC amplitude: -124.38 ± 47.71 pA, mean ± S.E.M.; n = 6; p < 0.025, paired t test) (Fig. 5, A–C). Together with the potency of L-AP4 and the lack of effect of (S)-3,4-DCPG, these data suggest that the L-AP4 modulation of synaptic transmission at the rat STN-SNc synapse is mediated by an activation of mGluR4.
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Group III mGluR-Mediate Modulation of Transmission in mGluR4 Knockout Mice. Our pharmacological studies suggest that mGluR4 modulates glutamatergic transmission in the SNc. In the attempt to provide further confirmation, we examined the group III mGluR-mediated modulation of excitatory transmission in wild-type and mGluR4 knockout mice. We first characterized dopamine neurons of 129X1/SvJ wild-type mice in current-clamp mode (Fig. 6A). Mouse neurons of the SNc exhibited electrophysiological properties consistent with dopaminergic neurons, including low-frequency spike firing, large afterhyperpolarizations, and a time-dependent inward rectification produced by the injection of hyperpolarizing currents.
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EPSCs were evoked by electrical stimulation identical to that used in the rat experiments (Fig. 6B). Consistent with our findings in the rat, L-AP4 inhibited excitatory transmission in the SNc of slices prepared from 129X1/SvJ mice. L-AP4 produced a maximal inhibition of transmission at a concentrations of 1 to 10 µM (predrug amplitude: -190.8 ± 45.84 pA, mean ± S.E.M.; 10 µM L-AP4 amplitude: -94.32 ± 25.46 pA, mean ± S.E.M.; n = 5; p < 0.05, paired t test) (Fig. 6, B and D). The effect was dose-dependent and reversible (Fig. 6, C and D).
Surprisingly, in mGluR4 knockout mice, the application of 10 µM L-AP4 produced a 62 ± 9% inhibition of the excitatory transmission (predrug amplitude: -130.48 ± 36.31 pA, mean ± S.E.M.; 10 µM L-AP4 amplitude: -50.94 ± 28.25 pA, mean ± S.E.M.; n = 5; p < 0.05, paired t test) (Fig. 7, A and E) that reversed with the drug washed (Fig. 7C). This suggests that another group III mGluR modulates excitatory transmission in the mGluR4 knockout mouse. Based on the high potency of this effect, mGluR8 is the most likely receptor to mediate inhibition of the EPSCs. Consistent with this, application of 300 nM (S)-3,4-DCPG produced a significant inhibition of excitatory transmission in the SNc in slices from mGluR4 knockout mice (predrug amplitude: -140.5 ± 40.98 pA, mean ± S.E.M.; 300 nM of (S)-3,4-DCPG amplitude: -78.25 ± 34.87 pA, mean ± S.E.M.; n = 4; p < 0.05, paired t test) (Fig. 7, B, D, and E).
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As observed in slices from rats, application of 30 µM PHCCC alone had no significant effect on excitatory synaptic transmission in the SNc (predrug amplitude: -110.5 ± 45.6 pA, mean ± S.E.M.; 30 µM PHCCC amplitude: -118.2 ± 46.4 pA, mean ± S.E.M.; n = 4; p > 0.05, paired t test). Coapplication of 30 µM PHCCC and a submaximal dose of L-AP4 (300 nM) produced a dramatic potentiation of L-AP4-induced inhibition of EPSC amplitude (predrug amplitude: -199.67 ± 39.54 pA, mean ± S.E.M.; 300 nM L-AP4 and 30 µM PHCCC amplitude: -115.87 ± 35.22 pA, mean ± S.E.M.; n = 6; p < 0.05, paired t test) when compared with 300 nM L-AP4 alone (predrug amplitude: -136.44 ± 15.02 pA, mean ± S.E.M.; 300 nM L-AP4 amplitude: -102.94 ± 14.71 pA, mean ± S.E.M.; n = 5; p < 0.05, paired t test) (Fig. 8, A–C), indicating that both mGluR8 and mGluR4 play a role in modulating the excitatory transmission in the SNc of mice. Interestingly, the effect of (S)-3,4-DCPG observed in mGluR4 knockout mice was significantly greater than the effect observed in the wild-type controls (ANOVA, Fisher's LSD, p < 0.05). Although this finding must be interpreted with caution, it raises the interesting possibility that a functional compensation occurs in response to the loss of mGluR4 in the knockout mouse.
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Discussion |
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Based on the high potency of L-AP4 in inhibiting excitatory transmission in the SNc, mGluR4 and mGluR8 are the most likely receptors to mediate the effects observed in these studies. The anatomical localization of mGluR4 mRNA in the rat (Testa et al., 1994; Messenger et al., 2002) and protein in both the rat and mouse (Bradley et al., 1999; Corti et al., 2002) has been previously described. Within the basal ganglia, mGluR4 immunoreactivity is observed at high levels in the globus pallidus, where it has been demonstrated to exist on presynaptic terminals that originate from the striatum (Bradley et al., 1999). In addition, there is a significant mGluR4-positive staining observed in the substantia nigra pars reticulata (Bradley et al., 1999; Corti et al., 2002). To date there has not been an immunocytochemical study that has reported the distribution of mGluR8 within the basal ganglia; however, in situ hybridization studies have found significant expression of both mGluR4 and mGluR8 in neurons of the rat STN (Testa et al., 1994; Messenger et al., 2002).
Since the STN represents the most likely source of excitatory afferents stimulated in these studies, either mGluR4 or mGluR8 may be appropriately expressed and trafficked to presynaptic STN terminals to mediate this response. Our pharmacological studies in rat strongly support the hypothesis that mGluR4 mediates the L-AP4-induced inhibition of excitatory transmission in the SNc. The lack of immunoreactivity in the SNc may be due to low but functional levels of mGluR4 that escape immunocytochemical detection. Alternatively, it is possible that the synapses stimulated in these studies are predominately outside the SNc. Dopamine neurons are known to send dendrites deep within the SNr (Iribe et al., 1999), and STN afferents are known to associate with and terminate on dopaminergic dendrites within the SNr (Smith et al., 1990). Therefore, the pattern of mGluR4 immunoreactivity is consistent with modulation of the STN-SNc synapse.
In the mouse, immunocytochemical studies have revealed a distribution of mGluR4 that is identical to that observed in the rat (Bradley et al., 1999; Corti et al., 2002). As stated above, no immunocytochemical studies are available to determine the localization of mGluR8 protein in the rodent central nervous system. However, a comparison of [3H]L-AP4 autoradiography between wild-type and mGluR4 knockout mice revealed approximately 20% specific binding remaining in the SNr of mGluR4-deficient mice (Thomsen and Hampson, 1999). This high-affinity specific binding is very likely due to the presence of mGluR8. Therefore, it is possible that both mGluR4 and mGluR8 protein is appropriately localized in the mouse to mediate the effects observed in the current studies. Future immunocytochemical studies will be necessary to confirm this hypothesis and to determine whether mGluR8 is also present in the rat SNr.
Although our present findings are consistent with what is known regarding the anatomical distribution of receptors in adult rats and mice, it is important to note that these studies have been performed in young animals. Recent work provides evidence for the developmental modulation of other mGluRs (Hubert and Smith, 2004). Although it is not currently known, it remains possible that the group III mGluRs are developmentally regulated in the basal ganglia. Our finding that there is no difference in the effect of (S)-3,4-DCPG in rats between 7- and 20-days postnatal suggests that there is not a gross developmental switch between mGluR8 and mGluR4; however, further anatomical studies are needed to better characterize changes in the expression of these receptors during development.
We previously have suggested that activation of mGluR4 may provide a novel palliative intervention for the treatment of PD (Valenti et al., 2003). From the standpoint of neurodegeneration associated with PD, the finding that mGluR4 activation also depresses glutamatergic transmission in the SNc has an interesting implication for this therapeutic strategy. The etiology of Parkinsonian neurodegeneration is complex and appears to involve a variety of factors, including mitochondrial dysfunction and oxidative stress (Beal, 2000; Zhang et al., 2000). In addition, an indirect excitotoxic hypothesis has been proposed to explain, in part, the degeneration of dopamine neurons in PD (Albin and Greenamyre, 1992). According to this hypothesis, an inhibition of normal mitochondrial function leads to an energy deficit and the subsequent inability of neurons to maintain a normal resting potential (Erecinska and Dagani, 1990). The resulting depolarization leads to a relief of the magnesium block of the N-methyl-D-aspartate receptors and a state in which normal levels of glutamate become toxic (Novelli et al., 1988). According to the current model of basal ganglia circuitry, the loss of striatal dopaminergic tone results in an excessive subthalamic glutamatergic drive that would be expected to exacerbate the neuronal loss associated with PD.
Consistent with this model, rodent studies have demonstrated that lesions of the STN decrease the 6-hydroxydopamine-induced degeneration of SNc neurons (Piallat et al., 1996). Furthermore, N-methyl-D-aspartate antagonists have been found to decrease the 1-methyl-4-phenylpyridinium-induced degeneration of SNc neurons (Turski et al., 1991) as well as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of SNc neurons in primates (Zuddas et al., 1992). Interestingly, direct application of (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) in the rat STN has been found to block the 6-hyrdoxydopamine-induced degeneration of SNc neurons (Blandini et al., 2001). Together, these studies suggest that a decrease in excitatory glutamatergic drive from the STN may be sufficient to provide some neuroprotective benefit in PD. This raises the exciting possibility that selective agonists acting at mGluR4 may provide both palliative benefits and a slowing of disease progression.
The apparent species difference observed in these experiments suggests that studies of dopamine receptor physiology and pathophysiology in different species must be interpreted with caution. For example, a brain penetrant mGluR4 agonist may provide both palliative and neuroprotective benefits in a rat model but not in the more commonly used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of neurodegeneration. Of course, the more important question of relevance to clinical PD must await a more detailed analysis of the distribution of these receptors in nonhuman primate and human brains.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: SNc, substantia nigra pars compacta; STN, subthalamic nucleus; PD, Parkinson's disease; mGluR, metabotropic glutamate receptor; CGP55845 (2S)-3-[[(1S)-1-(3,4-chlorophenyl)ethyl]amino-2-hydroxypropyl]phosphinic acid; CPPG, (R,S)-
-cyclopropyl-4-phosphonophenylglycine; (S)-3,4-DCPG, (S)-3,4-dicarboxyphenylglycine; PHCCC, N-phenyl-7-(hydroxymino)cyclopropa[b]chromen-1a-carboxamide; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; ACSF, artificial cerebrospinal fluid; EPSC, excitatory postsynaptic current; SNr, substantia nigra pars reticulata; ANOVA, analysis of variance; LSD, least significant difference; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline.
1 Current address: Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA. 
Address correspondence to: Dr. Michael J. Marino, Neuroscience Drug Discovery, Movement Disorders, Merck Research Laboratories, Merck and Co., Inc., WP46-300, 770 Sumneytown Pike, West Point, PA 19486. E-mail michael_marino{at}merck.com
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, significant difference between 129X1/SvJ and mGluR4 knockout, p < 0.05, ANOVA, Fisher's LSD.